Element, method and kit for the determination of an analyte in a liquid

ABSTRACT

The invention concerns an element for the determination of an analyte in a liquid by means of a specific binding reaction of two bioaffine binding partners containing in or on material which enables liquid transport between the zones. The element comprises a sample application zone; a detection zone located downstream thereof that is devoid of binding reagents and is the last zone on the element that allows liquid transport; a zone containing immobilized analyte or analyte analogue between the sample application zone and the detection zone; and impregnated conjugate that can be detached by liquid located in the sample application zone or upstream or downstream thereof composed of a bioaffine binding partner capable of a specific binding reaction with the analyte to be determined and a detectable label.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 of GermanApplication Serial No. 199 27 783.4 filed Jun. 18, 1999.

BACKGROUND AND SUMMARY OF THE INVENTION

The invention concerns an element for the determination of an analyte ina liquid by means of a specific binding reaction of two bioaffinebinding partners containing in or on material which enables liquidtransport between zones, a sample application zone and a detection zonelocated downstream thereof and a zone containing immobilized analyte oranalyte analogue between the sample application zone and detection zone,and impregnated conjugate that can be detached by liquid and is locatedin the sample application zone or upstream or downstream thereofcomposed of a bioaffine binding partner capable of a specific bindingreaction with the analyte to be determined and a detectable label.

Such elements are known for example from DE-A3842702 or DE-A4439429. Thedescribed analytical elements contain the necessary reagents to carryout immunoenzymometric or immunoenzymometric-like methods ofdetermination. In particular these are analyte or analyte analogueimmobilized in a zone, between the sample application zone and detectionzone and a conjugate composed of a bioaffine binding partner capable ofa specific binding reaction with the analyte to be determined and adetectable label.

In the case of DE-A 3842702 an enzyme label is described as thedetectable label. In order to make this label visible it is necessary tocontact it with a chromogenic enzyme substrate such that a colour isformed as a result of the enzymatic activity. The requirement that thelabel has to be made visible is a complication, costly due to themeasures that have to be taken and furthermore may result in technicaldifficulties for example when the corresponding enzyme substrate hasstability problems in the analytical element.

Therefore recently direct labels have been preferred as described inDE-A 4439429. These direct labels are for example metal or latexparticles which have an intrinsic colour and can be visualized with thenaked eye. Nowadays a gold label is particularly preferred. For this anappropriately labelled bioaffine binding partner depending on theanalyte is prepared for which optimal conditions then have to be createdon the analytical element for reaction and storage. This individualadaptation to the analyte to be determined is very laborious. Dependingon the analyte to be determined, the required polyclonal or monoclonalantibodies can behave very differently when conjugated to goldparticles. This can lead to different stabilities of gold conjugates.The differences in the behaviour of the various polyclonal antibodies orvarious monoclonal antibodies when coated on gold particles can resultin quite different spatial arrangements of the antibodies on the goldparticles which leads to steric problems when such conjugates arereacted with the analyte and can thus result in a poor sensitivity.

The object of the present invention was therefore to avoid thesedisadvantages and to provide an analytical element containing stablereagents that can be produced simply and reproducibly and which enablesa sensitive determination reaction.

This object is achieved by the subject matter of the invention asdescribed in the claims.

The invention concerns an element for the determination of an analyte ina liquid by means of a specific binding reaction of two bioaffinebinding partners

-   containing in or on material which enables liquid transport between    zones, a sample application zone and a detection zone located    downstream thereof-   as well as a zone containing immobilized analyte or analyte analogue    between the sample application zone and detection zone-   and an impregnated conjugate 1 upstream of the zone containing    immobilized analyte or analyte analogue that can be detached by    liquid and is composed of a bioaffine binding partner 1 capable of a    specific binding reaction with the analyte to be determined and a    detectable label 1,-   characterized in that-   the detectable label 1 is a low molecular organic molecule and a    universal conjugate 2 is present upstream of the zone containing    immobilized analyte or analyte analogue which can also be detached    by liquid and is composed of a bioaffine binding partner 2 capable    of a specific binding reaction with the detectable label 1 and a    visually detectable label 2.

The invention additionally concerns the use of an element according tothe invention to determine an analyte and a corresponding method ofdetermination. This method for the determination of an analyte by meansof an element according to the invention is characterized in that

-   a sample application zone is contacted with analyte,-   the analyte is moved by liquid towards the detection zone,-   analyte present in this liquid reacts with conjugates 1 and 2 to    form a detection complex,-   the detection complex is transported by liquid into the detection    zone-   and is determined there.

Finally the invention concerns a kit for the determination of an analytecontaining an analytical element according to the invention and anelution agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of an element according to the invention;

FIG. 2 is a top view of an element according to the invention;

FIG. 3 is a top view of an element according to the invention; and

FIG. 4 is a top view of an element according to the invention whichcontains two adjacent zones, which each carry a different one of twoconjugates.

DETAILED DESCRIPTION OF THE INVENTION

The determination of an analyte means of an element according to theinvention is based on a specific binding reaction of two bioaffinebinding partners. Bioaffine binding partners and corresponding specificbinding reactions between the binding partners are known to a personskilled in the art. Bioaffine binding partners are for example haptenand antibody, antigen and antibody, lectin and sugar or saccharide,avidin or streptavidin and biotin as well as nucleic acid and nucleicacid, ligand and receptor. In this case an antigen can be any moleculeagainst which it is experimentally possible to produce antibodies. Anantigen can also be an antibody or a particular site on an antibodywhich is referred to as an epitope and which can be specificallyrecognized and bound by an antibody. Nucleic acids are to be understoodas all possible forms of nucleic acids which are able to bind by meansof complementary bases. Special mention is made of DNA, RNA and alsonucleic acid analogues such as peptide nucleic acids (PNA, see forexample WO 92/20702) which is not to be regarded as a definitive list.Ligand and receptor refer quite generally to a specific bindinginteraction between two partners such as between a hormone and hormonereceptor.

The element according to the invention contains the reagents required tocarry out the determination of an analyte and other zones necessary forthe function of the element in or on material which enables a liquidtransport. It is important for the analytical element according to theinvention that liquid can move within the element towards the detectionzone. Such a liquid flow is for example possible in a suitably preparedhollow body by means of gravitational force. Devices which enable liquidtransport by centrifugal force as one form of gravitational force arefor example known from EP-B 0052769. However, analytical elementsaccording to the invention preferably contain absorbent materials thatcan move liquid by capillary force. The materials of the individualzones of the element according to the invention can be the same ordifferent. It will frequently be the case that different zones arecomposed of different materials if these are to optimally fulfil theirfunction.

Basically all materials are potentially suitable as absorbent capillaryactive materials which can be generally used for liquid uptake inso-called “dry tests” as described for example in U.S. Pat. No.4,861,711, U.S. Pat. No. 5,591,645 or EP-A 0291194 or in DE-A 3842702 orDE-A 4439429. For example porous materials have proven to beadvantageous for this purpose such as membranes e.g. nitrocellulosemembranes. However, it is also possible to use fibrous absorbent matrixmaterials such as fleeces, fabrics or knitted fabrics. Fleeces areparticularly preferred. Fibrous matrix materials can contain glass,cellulose, cellulose derivatives, polyester, polyamide and also viscose,synthetic wool and polyvinyl alcohol. Fleeces made of fibres based oncellulose, polymer fibres based on polyester and/or polyamide and anorganic binder as OH and/or ester groups as known from EP-B 0326135 canfor example be used in the invention. Fleece materials containingmeltable copolyester fibres in addition to glass fibres, polyesterfibres, polyamide fibres, cellulose fibres or cellulose derivativefibres as described in the European Patent Application 0571941 can alsobe used in analytical elements according to the invention. Papers suchas tea bag paper can also be readily used.

In order to improve the handling of the analytical element according tothe invention the absorbent capillary-active material or variousabsorbent capillary-active materials can be arranged on a rigid supportmaterial which in turn is not permeable to liquid, does not adverselyaffect the liquid flow in the matrix material and is inert with regardto the reactions which occur in the analytical element. A preferredsupport material can for example be a polyester foil onto which thematrix material enabling liquid transport is attached.

The individual zones in the element according to the invention can bearranged above one another, adjacent to one another, or partially aboveone another and partially adjacent to one another on the supportmaterial. An analytical element according to the invention isparticularly preferred in which the sample application zone, the zonecontaining immobilized analyte or analyte analogue and detection zoneare arranged adjacent to one another on the support material. In thisconnection adjacent to one another means that the zones are in directcontact with one another in the direction of liquid transport or areessentially disposed in one plane separated by other zones.

The sample application zone is the region of the element according tothe invention on which the sample is applied in which it is intended todetermine whether a particular analyte is present or, optionally, inwhich quantity it is present.

The detection zone is the region of the analytical element according tothe invention in which it is determined whether the examined analyte waspresent in the sample applied to the element. This determination can bequalitative, semi-quantitative or quantitative. In this connectionsemi-quantitative means that no definite concentration value isdetermined for the analyte but rather a concentration range in which theanalyte concentration is present.

According to the invention a zone containing immobilized analyte oranalyte analogue is located between the sample application zone anddetection zone. In this connection analyte analogue is understood as asubstance which behaves comparably to the analyte to be determined withregard to a specific binding reaction with the bioaffine bindingpartner.

The analyte or analyte analogue can be immobilized on a matrix materialbetween the sample application zone and detection zone by methods knownto a person skilled in the art. Thus it is for example possible toadsorb the analyte or analyte analogue onto a suitable matrix materialin such a manner that the analyte or analyte analogue is not detached byliquid under the test conditions. Of course the immobilization can alsobe by chemical means with formation of covalent bonds. An analyte can beimmobilized on the matrix material either directly or via a spacer. Inthe case of a spacer the analyte is usually chemically modified with asuitable spacer and then this analyte analogue is bound to the matrixmaterial. However, an indirect binding of the analyte or analyteanalogue to the matrix material can also be achieved by means of twobioaffine binding partners such as biotin and streptavidin.

Polyhaptens have proven to be particularly suitable as an analyteanalogue when detecting haptens. Polyhaptens are substances which have aplurality of haptens so that a plurality of bioaffine binding partnerscan be specifically bound thereto. Due to the high density of bioaffinebinding partners that can be achieved in this manner it is possible toattain a high test sensitivity.

The invention requires the presence of two conjugates on the element.These conjugates can be located in the sample application zone. They canalso be arranged upstream or downstream of the sample application zone.The conjugates can be impregnated in the suitable matrix material. Butthey can also be coated on the matrix material. Conjugates 1 and 2 canbe present as a mixture. But they can also be arranged separately andthen do not have to also necessarily be located directly next to oneanother. In the latter case it is then also possible that for exampleone conjugate is located upstream of the sample application zone and theother conjugate is for example located in a zone between the sampleapplication zone and the zone containing the immobilized analyte oranalyte analogue.

Conjugate 1 is composed of a bioaffine binding partner 1 capable of aspecific binding reaction with the analyte to be determined and adetectable label 1. If the analyte to be determined is a hapten or anantigen, the bioaffine binding partner 1 is an antibody which canundergo a specific binding reaction with the analyte. If the analyte isan antibody, the bioaffine binding partner 1 can be a correspondinghapten or antigen which can undergo a specific binding reaction with theantibody.

The detectable label 1 is understood according to the invention as a lowmolecular organic molecule, preferably an organic molecule with amolecular weight of less than 1,500, particularly preferably of lessthan 1,000 which as a hapten can be bound by an appropriate antibody ina specific binding reaction and can thus be detected. Digoxigenin ordigoxin have proven to be excellently suitable for this purpose. Digoxinis quite especially preferred.

The label 1 is preferably bound covalently to the bioaffine bindingpartner 1. Such conjugates can be reproducibly prepared by simpleorganic-chemical reactions.

Conjugate 2 is, depending on the analyte to be determined, a conjugatethat can be used universally composed of a bioaffine binding partner 2capable of a specific binding reaction with the detectable label 1 and avisually detectable label 2. The bioaffine binding partner 2 isparticularly preferably an antibody against the low molecular organicmolecule used as the label 1. So-called direct labels are preferablyused as the visually detectable label 2 i.e. labels which can berecognized by the eye as a result of their colour without furtherhandling steps. Advantageous labels of this type are for exampleparticles that are insoluble in water such as metal or latex particlesand also pigments such as silicate, carbon black or selenium. Inparticular metal particles are preferably used according to theinvention as a label. Colloidal gold is particularly preferred as alabel. The label 2 can be bound covalently or adsorptively to thebioaffine binding partner 2 whereby adsorptively includes allpossibilities apart from covalent binding. In the case of colloidalmetals as direct labels, in particular colloidal gold, adsorptive bondsare preferably utilized.

It is important for the invention that both conjugates are located in oron the matrix material upstream of the zone containing immobilizedanalyte or analyte analogue in such a way that they can be detached byliquid and transported towards the detection zone.

According to the invention both conjugates or only one of the conjugatescan be located upstream of the sample application zone in the elementaccording to the invention. In this case it is necessary that an elutionagent application zone is also located upstream of the sampleapplication zone. In this case it is necessary that after samplecontaining analyte has been applied to the sample application zone, theconjugate/the conjugates is/are transported by an additionally appliedelution agent through the sample application zone towards the detectionzone. In this case water or suitable aqueous solutions such as buffersare suitable as elution agents.

In an alternative embodiment of the analytical element according to theinvention an elution agent application zone separate from the sampleapplication zone is not provided. This is adequate when the sampleapplication zone is arranged upstream of the zone or zones containingthe conjugates, of the zone containing immobilized analyte or analyteanalogue and of the detection zone.

It is also possible that an elution agent application zone is locatedupstream of the sample application zone either on separate matrixmaterials or on the same matrix material.

Use of two conjugates as described above in an element according to theinvention for the determination of an analyte has considerableadvantages compared to the prior art embodiments. Thus the universalconjugate 2 is a stable conjugate composed of a visually detectablelabel and a bioaffine binding partner which has a high sensitivitytowards a low molecular organic molecule. Conjugate 2 can be producedreproducibly. The use of a low molecular organic molecule as adetectable label 1 enables polyclonal antibodies or monoclonalantibodies to be conjugated reproducibly, simply and in the same qualityby chemical methods. In this manner defined products are produced. Inimmunoassays with labelled binding partners of the analyte the labelledbinding partner is usually a critical component. Optimization workespecially with regard to the storage life of the components on theanalytical element and already before processing and the sensitivity ofthese components is not necessary with the present invention or is atleast considerably simplified. This also applies to the optimization ofreaction conditions on the analytical element and the ability to elutethe conjugate in the analytical element which can be largelystandardized. This work is mainly limited only to conjugate 1 which, dueto the nature of its components, can be much more simply optimized thana conjugate composed of a bioaffine binding partner which variesaccording to the analyte and a visually detectable label which are oftenboth very heterogeneous and cannot be determined exactly and thus withreference to the product are difficult to produce in the samereproducible quality.

An analyte is determined using an element according to the invention insuch a way that the sample which is to be examined for the presence ofanalyte is contacted with the sample application zone. Either theanalyte is itself already dissolved or suspended in liquid or additionalliquid is applied to the element as an elution agent in order totransport the constituents of the sample, especially the analyte whichmay be present, with liquid towards the detection zone. The analytepresent in the sample thereby comes into contact with the mixture ofconjugates 1 and 2 and reacts with them to form a detection complex.This detection complex is transported with the liquid into the detectionzone and is determined there. Only if analyte was present in the sampledoes visually detectable label 2 reach the detection zone in the form ofthe previously mentioned complex and can be detected there. If noanalyte was present in the examined sample, the mixture of conjugates 1and 2 is bound in the zone containing immobilized analyte or analyteanalogue and no visually detectable label 2 reaches the detection zone.In order to function optimally, conjugate 1 as well as conjugate 2 arepresent at such a concentration that the analyte or the analyte analogueimmobilized in the zone between the sample application zone anddetection zone is able to completely bind the conjugates. Conjugates andcorrespondingly also the immobilized analyte or the immobilized analyteanalogue should be present in an excess relative to the analyte to bedetermined. It is particularly preferred that conjugate 1 is present inan excess relative to conjugate 2 because a particularly highsensitivity is achieved. A six- to ten-fold excess of conjugate 1relative to conjugate 2 has proven to be particularly advantageous.

If it is intended to transport the sample through the element accordingto the invention into the detection zone using an additional elutionagent, a kit has proven to be advantageous which is composed of theanalytical element according to the invention and a correspondingelution agent. In this case the elution agent can be water or an aqueoussolution, preferably a buffer solution and the elution agent is in asuitable container. This container can for example be a dropping bottlein order to apply the liquid to the elution agent application zone. Itcan, however, also for example be a cup which is closed with a cap whennot in use and can be de-capped to carry out the method ofdetermination, and an element according to the invention can be placedin the cup containing the elution agent liquid in such a manner thatelution agent is taken up via the elution agent application zone andmigrates through the various zones into the detection zone.

A top-view of four different possible embodiments of an elementaccording to the invention is shown in FIGS. 1-4.

A particularly preferred embodiment of an element according to theinvention is shown in FIG. 1. Matrix materials (1–4) are attached sideby side on a rigid inert support foil (5) in such a manner that theirends butt or slightly overlap. The matrix materials (1–4) represent thetest zones of the analytical element according to the invention. Theyare preferably composed of different absorbent materials (papers,fleeces, porous plastic layers and such like) and liquid contact at theabutting edges is achieved by sufficiently close adjoining of thelayers. However, in an alternative embodiment it is of course alsopossible that several adjacent zones are made in one piece or severalpieces from the same material. Overall the test zones form a liquidtransport path which leads from the sample application zone (1) throughthe conjugate zone (2) containing conjugate 1 and conjugate 2 and thecapture zone (3) containing immobilized analyte or analyte analogue intothe detection zone (4). The conjugates 1 and 2 can be applied togetherin the conjugate zone (2) after prior mixing of suitable solutions orsuspensions.

The conjugate zone (2) can also be firstly impregnated with oneconjugate and then reimpregnated with the other conjugate. Or theconjugate zone (2) can contain two identical or different matrixmaterials lying on top of one another each of which carries a differentconjugate.

In the present case the analytical element is one in which eithersufficient liquid sample is applied that the liquid volume is adequateto supply all matrix materials including the detection zone (4) withliquid or in which firstly sample is applied to the sample applicationzone (1) which is then subsequently transported through the elementaccording to the invention by a special elution agent which is alsoapplied to the sample application zone (1). In the case of a samplecontaining analyte a detection complex composed of analyte andconjugates 1 and 2 is formed from conjugate zone (2) onwards as theanalyte is transported with liquid through the various zones (1–4). Thiscomplex migrates through the capture zone (3) and reaches the detectionzone (4) where for example a gold label serving as the label 2 isdetectable by eye as a red colouration. When the sample contains noanalyte, during liquid transport through the zones (1–4) of the elementaccording to the invention, the conjugates are transported from theconjugate zone (2) into the capture zone (3) where the conjugate mixtureis bound to the immobilized analyte or analyte analogue. Visuallydetectable label 2 does not reach the detection zone (4) in this case.No colouration will be detectable there.

Elements according to the invention are shown in FIGS. 2 and 3 in whichan elution agent application zone (6) is placed in front of the sampleapplication zone (1). When using such elements the sample is firstlyapplied to the sample application zone (1). Subsequently sufficientelution agent is applied to the elution agent application zone (6) thatanalyte is transported into the conjugate zone (2) where it can form acomplex with conjugates 1 and 2 and the complex that is formed reachesthe detection zone (4) via zone (3) containing immobilized analyte oranalyte analogue where the analyte is detected.

However, the elution agent application zone (6) of the elements of FIG.2 or 3 can be placed in so much elution agent that the sampleapplication zone (1) in the case of an element of FIG. 2 or theconjugate zone (2) in the case of an element of FIG. 3 is located abovethe liquid level of the elution agent.

Whereas in FIG. 2 the sequence of zones (1–4) is the same as in theelement of FIG. 1, the sequence of the sample application zone (1) andconjugate zone (2) in the element of FIG. 3 is interchanged. In thiscase the elution agent firstly comes into contact with conjugates 1 and2 and preincubates these before the analyte is contacted and bound.

FIG. 4 shows an element according to the invention which contains twoadjacent zones 2 a and 2 b which each carry a different one of the twoconjugates 1 and 2. In this case the conjugates can be located on thesame one-piece or two-piece matrix material or they can be present ondifferent types of matrix materials. The sequence of zones (1), (3) and(4) relative to one another and relative to the conjugate zone which isdivided into the partial regions 2 a and 2 b is identical to that shownin FIG. 1. The function of the element of FIG. 4 therefore correspondsto that described for the element of FIG. 1 except that the conjugates 1and 2 are dissolved successively.

The invention is elucidated in more detail by the following example.

EXAMPLE 1

Determination of benzodiazepine using an element according to FIG. 1.

A. Preparation of a Conjugate Composed of Gold and Monoclonal Antibodyto Digoxin

Two conjugates are prepared. Conjugate A contains gold particles with asize of approximately 40 nm, loaded with an antibody concentration of 2mg/l. Conjugate B contains gold particles with a size of ca. 20 nm,loaded with an antibody concentration of 10 mg/l.

Gold sol with an average particle diameter of ca. 40 nm and ca. 20 nmwas prepared according to the method of Frens (Frens, G., “Preparationof gold dispersions of varying particle size: Controlled nucleation forthe regulation of the particle size in monodisperse gold suspensions” inNature: Physical Science 241 (1973), 20–22) by reducing a 0.01 percentby weight tetrachloroauric acid solution with trisodium citrate whileboiling.

The antibody-gold conjugate preparation is based on the method of Roth,J. “The colloidal gold marker system for light and electron microscopiccytochemistry” in Bullock, G. R. and Petrusz, P., eds., “Techniques inImmunocytochemistry”, vol. 2, New York, Academic Press, 1983, 216–284.

After cooling the previously described gold sol solution to roomtemperature, the pH value of the gold sol is adjusted to pH 8.0 with 0.2M potassium carbonate solution. A dialysed solution of monoclonal IgGantibody to digoxin (source: Roche Diagnostics GmbH, Mannheim, Germany)was added to the gold sol. The volume ratio of IgG solution to colloidalgold solution was 1:10. After 30 minute stirring at room temperature thegold conjugate was saturated by adding a highly concentrated bovineserum albumin solution (final concentration in the conjugate solution: 1mg/ml).

The gold conjugate was concentrated to an optical density of typically20 (absorbance at 525 nm and 1 cm light path) by ultrafiltration againsta 20 mM Tris buffer pH 8.0. The conjugate solution was finally adjustedto a final concentration of 100 μM Brij® and 0.05 percent by weightsodium azide.

B. Preparation of a Conjugate Composed of Digoxin and PolyclonalAntibody to Benzodiazepine

Sheep are immunized as described in example 1 of EP-A 0726275 with7-chloro-3-[2-(N-maleinimido)ethyl]oxy-1-methyl-5-phenyl-1H-1,4-benzodiazepine-2-(3H)-onethe synthesis of which is known from example 3 of EP-A 0726275. 100 mlserum of a sheep immunized in this manner containing ca. 6.5 g proteinwas firstly treated with 1.5 g aerosil (1 hour at room temperature) andcentrifuged. The supernatant was decanted and adjusted with ammoniumsulfate to 1.9 M. This precipitates the IgG. The precipitate wascentrifuged and the supernatant was decanted. The precipitate was takenup in weak PBS buffer pH 7 and dialysed. The dialysate was negativelypurified over 100 ml DEAE Sephadex ff. In this process impuritiesremained on the column and the immunoglobulin ran through. The eluatewas collected (detection at 280 nm) and rebuffered in PBS buffer pH 7.4for the immunosorption. Yield 3.5 g IgG in 90 ml solution.

3.5 g IgG in 90 ml from the DEAE purification was circulated by pumpover 50 ml immunoadsorber (Spherosil, to which a polyhapten composed ofTemazepam on rabbit IgG (see example 1D) is bound). In this process theimmunospecific IgGs were bound and all other proteins ran through. Itwas re-washed with PBS buffer. The bound IgG was washed further with twocolumn volumes of sodium chloride/Tween 20 and two column volumes of 30mM sodium chloride. Then it was eluted stepwise. The first step iselution with 3 mM hydrochloric acid at 4 to 8° C., the second step iselution with 1 M propionic acid at 4 to 8° C. and the third step iselution with 1 M propionic acid at room temperature. The eluates wereimmediately dialysed against ice water. The eluates were additionallydialysed against 1 mM acetic acid and, after concentration, they werefiltered and lyophilized. The yield was approximately 300 mg polyclonalIgG antibody to benzodiazepine.

The polyclonal antibodies prepared in this manner were dissolved inwater to a concentration to 12 mg antibody per ml. They were re-bufferedwith 1.5 ml 1 M potassium phosphate buffer pH 8.3 to 0.1 M potassiumphosphate and adjusted to 10 mg antibody per ml. Digoxin succinimideester was dissolved in DMSO to a concentration of 10 mg/ml and a 6-foldmolar excess of the digoxin derivative was pipetted at 4° C. into theantibody solution. It was allowed to react for three hours at 4° C.Subsequently it was dialysed against a 50-fold volume of 20 mM Tris pH 8and against a 50-fold volume of 50 mM sodium chloride for at least fourhours in each case.

C. Preparation of Polymerized Streptavidin

Polymerized streptavidin was prepared as described in examples 1 c and 1d of EP-B 0331127.

D. Preparation of Biotinylated Temazepam Polyhapten

1.2 g unspecific lyophilized polyclonal rabbit IgG was dissolved in 40ml potassium phosphate buffer pH 8.5 and after centrifugation the clearsupernatant was decanted. The supernatant contained 950 mg protein. 12.4mg S-acetylthiopropionic acid succininimidyl ester dissolved in 1.24 mlDMSO was added to the rabbit IgG solution and stirred for two hours atroom temperature. The reaction was stopped by addition of 0.5 ml 1 Mlysine solution and dialysed against triethylammonium citrate/EDTAsolution pH 6.5.

After dialysis the activated rabbit immunoglobulin was deacetylated fortwo hours at 4° C. with 1 ml 1 M ammonium hydroxide solution per 25 mlimmunoglobulin solution at pH 6.5.

For 950 mg deacetylated activated rabbit IgG in 50 ml buffer, 10.8 mgTemazepam (prepared according to example 3 in EP-A 0726275) wasdissolved in 1.08 ml DMSO and added to the deacetylated activated rabbitimmunoglobulin solution. After two hours at 4° C. the reaction wasstopped with 2 ml 0.1 M cysteine and the solution was incubated for 30minutes with 2 ml 0.5 M iodoacetamide and subsequently dialysed for 30minutes at room temperature against triethanolamine buffer pH 8.5.

24 mg biotin-succinimidyl ester dissolved in 2.4 ml DMSO was added to950 mg of the Temazepam polyhapten prepared in this manner and stirredfor two hours at room temperature. Subsequently it was intensivelydialysed against weak acetic acid (10 to 1 mM). After dialysis thedialysate was adjusted with 1 M sodium acetate pH 4.2 to 20 mM sodiumacetate pH 4.2 and purified by column chromatography overtris-acryl-carboxymethylcellulose.

E. Preparation of Analytical Elements According to FIG. 1

Two analytical elements according to the invention were prepared withdifferent conjugate zones (2).

The following zones according to FIG. 1 were glued onto support foil (5)of 5 mm width.

Sample application zone (1): Polyester fleece from the Binzer Company,Hatzfeld, Germany. A pure polyester fleece which is strengthened with10% Kuralon, has a thickness of 1.0 to 1.2 mm and an absorptive capacityof 1800 ml/m².

Conjugate zone (2): A mixed fleece composed of 80 parts polyester and 20parts synthetic wool strengthened with 20 parts Kuralon having athickness of 0.32 mm and an absorptive capacity of 500 ml/m² wasimpregnated with one of the following solutions and dried:

-   -   Impregnation solution A    -   A mixture of 1 ml of the gold conjugate A prepared in “A”        diluted in Hepes buffer (200 mM, pH 7.5) to an antibody        concentration of 0.3 nmol/ml and 1 ml of the digoxin conjugate        prepared in “B” diluted in Hepes buffer (200 mM, pH 7.5) to a        digoxin concentration of 2 nmol/ml is incubated for one hour at        room temperature. Subsequently it is impregnated in the fleece.    -   Impregnation solution B    -   A mixture of 1 ml of the gold conjugate B prepared in “A”        diluted in Hepes buffer (200 mM, pH 7.5) to an antibody        concentration of 1.4 nmol/ml and 1 ml of the digoxin conjugate        prepared in “B” diluted in Hepes buffer (200 mM, pH 7.5) to a        digoxin concentration of 2 nmol/ml is incubated for one hour at        room temperature. Subsequently it is impregnated in the fleece.

Capture zone (3): A fleece composed of 100% linters, strengthened withtwo percent by weight Etadurin with a thickness of 0.41 mm and anabsorptive capacity of 386 ml/m² is impregnated with a solution of 200mg/l of the polymerized streptavidin prepared in C in 50 mmol/l sodiumphosphate pH 8.0 and subsequently dried. The preimpregnated fleece issubsequently impregnated again with a solution of 300 mg/l of thebiotinylated Temazepam polyhapten prepared in D. in 50 mmol/l sodiumphosphate pH 8.0 and dried.

Detection zone (4): A fleece of 100% linters, strengthened with twopercent by weight Etadurin with a thickness of 0.35 mm and an absorptivecapacity of 372 ml/m² is used.

F. Test Procedure

In order to determine benzodiazepine with an element prepared in “E”,the test strip is dipped for ca. 5 seconds in the liquid to be examinedsuch that the sample application zone (1) is located about threequarters below the liquid level. Afterwards the element is placedhorizontally on a non-absorbent support. When the liquid front hascompletely wetted the detection zone (4) (usually after two minutes atmost in the case of aqueous solutions) a pink colouration indicates thepresence of benzodiazepines in the sample to be examined. When nosubstances are present which are recognized by the digoxin-labelledantibody the detection field remains white. The intensity of the pinkcolour correlates with the analyte concentration. A colour tablesimplifies the assignment.

-   colour field (FF)0: negative, no analyte-   colour field (FF)1: positive, light pink-   colour field (FF)2: strongly positive, strong red colour

Results table Aqueous solution containing Urine containing BromazepamBromazepam 0 ng/ml 100 ng/ml 0 ng/ml 50 ng/ml 100 ng/ml gold conjugate AFF 0 FF 1–2 FF 0 FF 0–1 FF 1–2 gold conjugate B FF 0 FF 2 — — —

1. An element for the determination of an analyte in a liquid, the element comprising: a sample application zone, a detection zone located downstream from the sample application zone and being the last zone of the element that allows liquid transport, the detection zone being devoid of a binding reagent that would enable detection of the analyte; a zone containing immobilized analyte or analyte analogue located between the sample application zone and the detection zone, a conjugate impregnated in a matrix material located upstream of the zone containing immobilized analyte or analyte analogue, the conjugate can be detached from the matrix material by liquid and comprises a first bioaffine binding partner capable of a specific binding reaction with the analyte to be determined and a first detectable label, wherein the first detectable label is a low molecular organic molecule, and a universal conjugate, located upstream of the zone containing immobilized analyte or analyte analogue, which can be detached by liquid and comprises a second bioaffine binding partner and a visually detectable label, the second bioaffine binding partner is capable of a specific binding reaction with the first detectable label, wherein the visually detectable label is a direct visually detectable label formed to carry out the determination of the analyte in the detection zone; wherein the matrix and the zones are made from liquid permeable transport materials.
 2. The element as claimed in claim 1, wherein the first detectable label is digoxigenin or digoxin.
 3. The element as claimed in claim 2, wherein the second bioaffine binding partner is an antibody to digoxigenin or digoxin.
 4. The element as claimed in claim 2, further comprising an elution agent application zone located upstream of the sample application zone.
 5. The element as claimed in claim 4, wherein the impregnated conjugate and the universal conjugate are located between the elution agent application zone and the sample application zone.
 6. The element as claimed in claim 1, wherein the second bioaffine binding partner is an antibody to digoxigenin or digoxin.
 7. The element as claimed in claim 1, wherein the visually detectable label is metal particles or latex particles.
 8. The element as claimed in claim 7, wherein the visually detectable label is gold particles.
 9. The element as claimed in claim 1, wherein the impregnated conjugate and the universal conjugate are located in the sample application zone.
 10. The element as claimed in claim 1, further comprising an elution agent application zone located upstream of the sample application zone.
 11. The element as claimed in claim 10, wherein the impregnated conjugate and the universal conjugate are located between the elution agent application zone and the sample application zone.
 12. The element as claimed in claim 10, wherein the impregnated conjugate and the universal conjugate are located in the sample application zone. 